脂质介质,其中包括专门的促分辨介体和经典的二十烷酸,是启动和解决炎症的关键。这些分子的调节决定了炎症是自然消退还是持续消退。然而,我们对这些介质在各种炎症环境中如何随时间调节的理解是有限的.这种差距阻碍了我们对疾病发作和进展的潜在机制的掌握。由于它们在许多组织中的局部作用和低内源性水平,开发稳健和高度敏感的方法对于评估不同炎症环境中的内源性调节至关重要.这些方法将帮助我们深入了解它们的生理作用。这里,我们建立了提取的方法,识别,量化这些中介。使用我们的方法,我们共鉴定出37种脂质介质.此外,通过使用反相HPLC方法,我们成功地分离了选择脂质介质的双键和手性异构体,包括脂氧素(LX)A4,15-epi-LXA4,保护素(PD)D1,PDX,和17R-PD1。在溶剂和替代基质中对标准曲线的线性进行方法验证,定量下限(LLOQ),准确度,和精度。来自这些研究的结果表明,线性良好,r2值>0.98,并且介质的LLOQ在相位上为0.01至0.9pg,在替代基质中为0.1至8.5pg。溶剂中日间和日内精密度的相对标准偏差(RSD)在5%到12%的范围内,中间,和高浓度,而在低至高浓度下,日内和日内变异性的准确度RSD范围为95%至87%。所用内标在生物基质(血浆和血清)中的回收率为60%至118%。我们观察到在负电离模式下评估的分子的明显离子抑制,而在正离子模式下评估的分子的基质具有离子增强作用。积分算法的比较,即,AutoPeak和MQ4,以及计算信噪比的方法(即,美国药典,相对噪声,峰峰之间,和标准偏差)证明了测试的不同积分算法对信噪比计算的影响很小。相比之下,用于计算信噪比的方法对结果有更显著的影响,相对噪声方法被证明是最稳健的。本文所述的方法提供了研究生物组织中的SPM和经典类二十烷酸的平台,这将有助于进一步理解疾病机制。
Lipid mediators, which include specialized pro-resolving mediators and classic eicosanoids, are pivotal in both initiating and resolving inflammation. The regulation of these molecules determines whether inflammation resolves naturally or persists. However, our understanding of how these mediators are regulated over time in various inflammatory contexts is limited. This gap hinders our grasp of the mechanisms underlying the disease onset and progression. Due to their localized action and low endogenous levels in many tissues, developing robust and highly sensitive methodologies is imperative for assessing their endogenous regulation in diverse inflammatory settings. These methodologies will help us gain insight into their physiological roles. Here, we establish methodologies for extracting, identifying, and quantifying these mediators. Using our methods, we identified a total of 37 lipid mediators. Additionally, by employing a reverse-phase HPLC method, we successfully separated both double-bond and chiral isomers of select lipid mediators, including Lipoxin (LX) A4, 15-epi-LXA4, Protectin (PD) D1, PDX, and 17R-PD1. Validation of the method was performed in both solvent and surrogate matrix for linearity of the standard curves, lower limits of quantitation (LLOQ), accuracy, and precision. Results from these studies demonstrated that linearity was good with r2 values > 0.98, and LLOQ for the mediators ranged from 0.01 to 0.9 pg in phase and from 0.1 to 8.5 pg in surrogate matrix. The relative standard deviation (RSD) for inter- and intraday precision in solvent ranged from 5% to 12% at low, intermediate, and high concentrations, whereas the RSD for the inter- and intraday variability in the accuracy ranged from 95% to 87% at low to high concentrations. The recovery in biological matrices (plasma and serum) for the internal standards used ranged from 60% to 118%. We observed a marked ion suppression for molecules evaluated in negative ionization mode, while there was an ion enhancement effect by the matrix for molecules evaluated in positive ionization mode. Comparison of the integration algorithms, namely, AutoPeak and MQ4, and approaches for calculating signal-to-noise ratios (i.e., US Pharmacopeia, relative noise, peak to peak, and standard deviation) demonstrated that different integration algorithms tested had little influence on signal-to-noise ratio calculations. In contrast, the method used to calculate the signal-to-noise ratio had a more significant effect on the results, with the relative noise approach proving to be the most robust. The methods described herein provide a platform to study the SPM and classic eicosanoids in biological tissues that will help further our understanding of disease mechanisms.